methods Flashcards
not all cells can be grown in a lab, they must be grown in their
unique happy places
affinity chromatography
protein has a special feature that sticks to the resin (agarose beads with antibodies or metals)
most selective
ion exchange chromatography
resin is charged and is attracting charged residues on proteins of interest
separation is achieved by changes of pH which releases bound proteins
hydrophobic interaction chromatography
resin is not charged and is attracting non-charged residues on proteins of interest
salt exposes hydrophobic residues of proteins
separation is achieved by changing [salt] –> water is now available (water is polar) –>
size exclusion chromatography
in resin (beads), there are little channels that allow proteins to flow through
big proteins flow through faster (because they avoid channels – small proteins flow through channels)
least selective
b cells –> plasma cells–>
antibodies
ELISA
enzyme-linked immunosorbent assay
an antibody binds an antigen of interest and produces a detectable signal
epitope
part of a large antigen that interacts with the antibody
not recognized when protein is unfolded
direct ELISA
a single Ab binds the antigen and carries the signal
indirect ELISA
primary Ab (usually polyclonal) binds the antigen; secondary Ab binds the primary Ab and carries the signal
sandwich ELISA
both Ab bind the antigen at unique epitopes
Order of addition:
- capture Ab is absorbed to the well
- antigen is added
- primary Ab is added –> binds to antigen and carries signal
competition/inhibition ELISA
antigen presence is indicated by an absence or decrease of signal
“if antigen is present is sample, we lose the signal”
“positive result is lack of signal”
Order of addition:
- antigen is added
- primary Ab + sample
- secondary Ab
ChIP (Chromsome Immunoprecipitation)
- formaldehyde covalently connects DNA and protein at the location of a non-covalent interaction (cross-link)
- the antibody used is specific to a DNA-binding protein
- collects all DNA crosslinked to the protein (collects all directly and indirectly linked DNA sequences)
- reversing the covalent bonds with formaldehyde releases the DNA
- DNA can be analyzed
agglutination
when blood typing, the interaction of antigen and antibody – linking of blood cells together
to avoid agglutination, Rh- mothers are given
RhoGAM (anti-D) to prevent the production of maternal antibodies against fetal Rho/D antigen
